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Isolation of Cryptococcus Neoformans and Other Opportunistic Fungi from Pigeon Droppings Rticle Maryam Soltani, Mansour Bayat, Seyed J

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Isolation of Cryptococcus Neoformans and Other Opportunistic Fungi from Pigeon Droppings Rticle Maryam Soltani, Mansour Bayat, Seyed J Isolation of Cryptococcus neoformans and other opportunistic fungi from pigeon droppings RTICLE Maryam Soltani, Mansour Bayat, Seyed J. Hashemi1, Mohammadali Zia2, Nader Pestechian3 A Department of Medical and Veterinary Mycology, Faculty of Specialized Veterinary Science, Science and Research Branch,1Department of Medical Mycology, School of Public Health Research, Tehran University of Medical Sciences, Tehran, 2Department of Basic Sciences, Khorasgan (Isfahan) Branch, Islamic Azad University, 3Department of Mycology and Parasitolog, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran Background: Invasive fungal infections cause considerable morbidity and mortality in immunocompromised hosts. Pigeon droppings could especially be a potential carrier in the spread of pathogenic yeasts and mold fungi into the environment. The objective of this RIGINAL study was to isolation of Cryptococcus neoformans and other opportunistic fungi from pigeon droppings. Materials and Methods: One hundred twenty samples of pigeon droppings were suspended 1:10 in saline solution and then cultured. Identification of C. neoformans O was performed on bird seed agar, presence of a capsule on India ink preparation, urease production on urea agar medium and RapID yeast plus system. The identification of candida species was based on micro‑morphological analysis on corn meal‑Tween 80 agar, RapID yeast plus system and growth in CHROMagar candida. The identification of other fungi was based on macromorphologic, microscopic, biochemical and physiological characteristics. Results: The highest frequency of yeasts and mold fungi were observed in Candida albicans 6.6% and Penicillium spp. 25%. The frequency rate of C. neoformans isolation was 2.5%. Conclusion: Several types of fungi are present in pigeon droppings that can spread in environment and transmit to children and elderly as well as immunocompromised patients who are at increased risk of contracting opportunistic diseases. Key words: Cryptococcus neoformans, candida albicans, immunocompromised hosts, opportunistic fungi, pigeon droppings INTRODUCTION region; this may be particularly true for mycoses that are acquired from the environment.[1] Immunocompromised patients are susceptible to bacterial, fungal, and viral infections that healthy Avian droppings have been reported as a potential immune systems usually conquer. They are also environmental source of human pathogenic yeasts. susceptible to common infections of childhood. The Pigeon droppings, which are abundant in public patient is immunocompromised either because of an areas, could especially be a potential carrier in the acquired or inherited immune deficiency disorder. The spread of pathogenic yeasts i‑nto the environment and [5] number of immunocompromised patients is increasing subsequently humans. due to the intensive therapy being administered those Since the 1960s a number of new species of yeast with cancer, organ transplant, and HIV infection.[1] has been reported as pathogens for humans because of the increase in immunodeficiency syndromes The incidence of invasive opportunistic mycoses has and immunosuppressant chemotherapy in cancer increased because of the expanding population of treatment.[6] immunosuppressed patients, including solid‑organ transplant (SOT) and hematopoietic stem cell Cryptococcus is a systemic infection caused by the transplant (HSCT) recipients, patients with cancer, yeast Cryptococcus neoformans, is the most common patients with AIDS, premature neonates, elderly life‑threatening systemic fungal infection occurring patients, and patients recovering from major surgery. in HIV‑infected patients.[4,5,7] Cryptococcus neoformans Despite some effective treatment options, such mycoses is an opportunistic fungal pathogen that causes are associated with high morbidity and mortality meningoencephalitis as the most frequent clinical [2‑4] rates. presentation in immunocompromised patients, mainly in people infected by HIV.[8] Opportunistic mycoses show distinct regional incidence patterns throughout the world and may exhibit different Yeasts of the genus Candida are those most epidemiologic features, depending on the geographic frequently isolated from blood stream infections in Address for correspondence: Dr. Maryam Soltani, Department of Medical and Veterinary Mycology, Faculty of Specialized Veterinary Science, Science and Research Branch, Islamic Azad University, Tehran, Iran. E‑mail: [email protected] Received: 14‑08‑2012; Revised: 18‑12‑2012; Accepted: 25‑12‑2012 | January 2013 | Journal of Research in Medical Sciences 56 Soltani, et al.: Isolation of Cryptococcus neoformans from pigeon droppings immunocompromised patients although they are also found determined by color changes of chromogenic substrates in the transient normal flora of the skin and digestive tract within the sample wells, some of which require addition of humans. Other important yeasts such as Rhodotorula, of reagents. Test reactions are grouped in triads and scored Trichosporon, were also frequently isolated from pigeon on the basis of a positive or negative reaction, resulting in excreta and the cloacae of coots respectively. These fungi can a 6‑digit microcode which is matched to an empirically cause blood infections in patients with indwelling central derived database of commonly occurring microcodes for venous catheters and cancer.[6] identification of the isolate.[17] In recent years, opportunistic fungal infections have The identification of candida species was based on increased substantially, and the species of the genus micro‑morphological analysis on corn meal‑Tween 80 Aspergillus, Penicillium, Mucor, Rhizopus, Paecilomyces, agar, RapID yeast plus system and growth in CHROMagar Fusarium, Alternaria, and Cladosporium are emerging as the candida.[18] cause of a variety of infections in humans.[9‑13] For each isolate, the identifications obtained to the species Large numbers of pigeons have inhabited different areas and genus level, but sometimes additional tests (low surrounding the hospital and can act as a source of probability identifications) were required to (i) distinguish opportunistic fungi, therefore hospitalized patients and between two or more possible species, (ii) for its discrepant immunocompromised persons are susceptible to a number identifications, and (iii) for its failure to provide and of opportunistic infections.[8] Thus it is important for these identifications (no codes). When the six‑digit microcode individuals to have an understanding of the risks associated provided an identification with a low percentage of with the different species of animals and birds. probability (<94%), the additional tests were performed by conventional methods.[19] The identification of other fungi The isolation and identification of different fungi from was based on macromorphologic, microscopic, biochemical pigeon droppings can provide useful information for and physiological characteristics. ecological and epidemiological studies. Therefore, we decided to survey about the presence of C. neoformans, RESULTS other yeasts and molds in pigeon droppings collected from pigeon towers. Three out of 120 samples, (2.5%), were diagnosed as Cryptococcus neoformans by using RapID yeast plus system MATERIALS AND METHODS and CGB test. One hundred twenty samples, each containing about 500 Isolated yeast species, other than Cryptococcus, were Candida grams of pigeon droppings were collected from 19 pigeon albicans (n = 8), C. tropicalis (n = 5), C. parapsilosis (n = 5), towers in urban areas of Isfahan in a period of 9 months (Sept. C. guilliermondii (n = 4), Rhodotorula spp. (n = 4) and 2010‑May 2011) and the samples were processed using a Trichosporon (n = 1). Mold fungi including Penicillium standard protocol.[14] In brief, a suspension of droppings, was spp. (n = 30), Aspergillus spp. (n = 25), Mucor spp. (n = 18), spread onto the surface on Sabouraud’s Dextrose Agar (SDA) Rhizopus spp. (n = 14), Alternaria spp. (n = 1), Fusarium medium supplemented with chloramphenicol (200 mg/l) spp. (n = 4), Cladosporium spp. (n = 2), Paecilomyces and incubated at 32°C for 15 days.[14] The identification spp. (n = 11) [Table 1]. of isolates was established by niger seed agar (NSA) (for melanin production) medium and India ink preparation.[15] The RapID yeast plus system is easy to use with only a Standard strain of C. neoformans ATCC 28957 was used as a few exceptions and the reactions are distinct and easy to positive control in parallel with samples. interpret. As can be seen in Table 2, 26 of 30 strains (86.66%) were correctly identified to the species level without Since C. neoformans is sensitive to canavanine and cannot additional tests. Three organisms (10%) yielded the correct utilize glycine as a sole carbon and nitrogen sources, and identification among two or more possibilities, with the will therefore not grow, and the media remains yellow correct identification upon performance of additional in color, therefore, biotyping was carried out using tests. These comprised Rhodotorula rubra with Trichosporon Canavanine‑Glycine‑Bromothymol blue (CGB) agar growth beigelii and Rhodotorula rubra, Candida guilliermondii with test, as described by Kwon Chung et al.[16] Additionally, C. guilliermondii and Candida famata and finallyCryptococcus cryptococcus identification was carried out by RapID yeast neoformans with C. neoformans and Pichia spp. Isolates [Table 3]. plus system (RYP) which is a 4‑h micro panel system that utilizes
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